Name: direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway
Uploaded: 2015-01-20T15:00:00
Description: Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Financiamento: Este trabalho foi apoiado por uma bolsa da Fundação Bertarelli (DJL), uma subvenção MED-EL (DJL), e um dos Institutos Nacionais de Saúde Grants DC01089 (MCB).

NOTA: Todos os procedimentos experimentais são realizados de acordo com o Cuidado e Uso Comitê Animal do Massachusetts Eye e Ear Infirmary e Harvard Medical School, que seguem as diretrizes nacionais de cuidados de animais, incluindo a Política Pública de Saúde de serviço na Humane Cuidado e Uso de Animais de Laboratório, o Guia ILAR, e da Lei do Bem-Estar Animal. Procedimentos experimentais listados abaixo exposição do DCN esquerda detalhe. Usar instrumentos esterilizados durante a realização da cirurgia de sobrevivência. <...

<ol>
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Este artigo descreve a técnica de visualização direta do DCN no modelo murino para a manipulação do sistema auditivo central. A abordagem descrita de visualização direta oferece vantagens significativas sobre a alternativa principal, que são abordagens estereotáxica. Primeiramente, a visualização direta da DCN permite confirmação imediata do local do tronco cerebral, enquanto abordagens estereotáxica não pagar a visualização direta. Em experiências que implicam o prolongados períodos de incubação, c...

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

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			<td height="21" style="height:21px;width:197px;">Stereotaxic holder</td>
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			<td height="51" rowspan="2" style="height:52px;width:197px;">10 &#181;l Hamilton syringe</td>
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			<td height="42" rowspan="2" style="height:42px;width:197px;">34 gauge, needle</td>
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			<td height="21" style="height:21px;">Rongeurs</td>
			<td>Fine Surgical Tools</td>
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direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

A investigação sobre o uso da transferência de genes mediada por vírus para prender ou reverter a perda auditiva tem sido amplamente relegado para o sistema auditivo periférico. Poucos estudos examinaram transferência de genes para o sistema auditivo central. O núcleo dorsal coclear (DCN) do tronco cerebral, que contém neurônios de segunda ordem da via auditiva, é um local potencial de transferência de genes. Neste protocolo, uma técnica para exposição direta e máxima da DCN murino através de uma abordagem posterior fossa é demonstrada. Esta abordagem permite a qualquer cirurgia aguda ou sobrevivência. Após a visualização direta da DCN, uma série de experimentos são possíveis, incluindo a injeção de opsinas para o núcleo coclear e subsequente estimulação por uma fibra óptica acoplada a uma luz de laser azul. Outros experimentos neurofisiologia, como a estimulação elétrica e traçados injetores neurais também são viáveis. O nível de Visualização e a duração da estimulação realizável que esta abordagem seja aplicável a uma ampla gama de experiências.

Parcial Cerebellar aspiração demonstra ter acesso ao núcleo coclear Após a pele e músculo sobrejacente ao crânio são removidos, marcadores de superfície do crânio, como as linhas de sutura coronal e lamda, demonstrar a localização aproximada da craniotomia. Após craniotomia com fórceps, o cerebelo é visualizada. Aspiração cuidadosa da pequena porção de o cerebelo demonstra visualização do CN, que podem então ser injectados (Figura 1). <p cla...

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

Visualização direta do Murino Dorsal Coclear Nucleus para Optogenetic Estimulação da via auditiva

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

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